This invention relates generally to antigenic preparations and more specifically to a Leptospira outer membrane protein (LipL32) which is useful for inducing a protective immune response in a subject.
Spirochetes are helically shaped, motile bacteria. Among the spirochetes, three genera are identified as pathogens, Treponema, Borrelia and Leptospira. The treponemes include Treponema pallidum, the causative agent of the venereal disease syphilis in humans. Other treponemes such as T. denticola and T. vincentii are usually found in the mouth and have been linked to periodontal disease. Several other spirochetes can cause animal as well as human disease. Borrelia recurrentis, which is carried by ticks, causes relapsing fever in which the infected subject suffers repeated bouts of fever and chills. B. burgdorferi, also tick-borne, is responsible for Lyme disease, a debilitating ailment known to cause arthritis, loss of myelin from nerve cells and myocarditis. Pathogenic strains of Leptospira frequently cause the zoonotic disease, leptospirosis, and are capable of infecting most mammalian species. At present, there are six pathogenic species and three nonpathogenic species within the genus Leptospira. Infection occurs either through direct contact with an infected animal or indirect contact with contaminated soil or water. In livestock, the disease causes economic losses due to abortion, stillbirth, infertility, decreased milk production, and death.
Efforts to control leptospirosis have been hampered because virulent leptospires have the capacity for both long-term survival in the environment as well as persistent infection and shedding by wildlife and livestock. Currently available leptospiral vaccines produce short-term immunity and do not provide cross-protection against many of the 170 serovars of pathogenic Leptospira (Thiermann, et al., J.Am.Vet.Med.Assoc. 184:722, 1984). These vaccines consist of inactivated whole organisms or outer envelope preparations which produce seroreactivity as determined by microscopic agglutination of intact organisms. The nature of the protective immunogens in these vaccine preparations has not been conclusively elucidated, although several lines of evidence suggest that lipopolysaccharide may confer a degree of serovar-specific protection.
The pathogenesis of leptospirosis is similar to that of other spirochetal diseases, including syphilis (caused by Treponema pallidum) and Lyme borreliosis (caused by Borrelia burgdorferi). Both syphilis and Lyme borreliosis are characterized by widespread dissemination early in the course of disease, including invasion of the central nervous system. Leptospira share this ability with other pathogenic spirochetes such that meningitis is a common manifestation of leptospirosis. Another feature of spirochetal infections is the ability to persist chronically in the host, as manifested in cases of tertiary syphilis and chronic Lyme arthritis.
Attempts to identify leptospiral outer membrane proteins (OMPs), have led to limited success due to such problems as: 1) the techniques used to identify surface-exposed proteins probably involved damage to the fragile leptospiral outer membrane resulting in exposure of subsurface structures; 2) putative surface-exposed proteins that were identified included a 35-36 kD doublet corresponding to Leptospira endoflagella (Kelson, et al., J. Med. Microbiol. 26:47, 1988), which are subsurface structures in spirochetes; and 3) use of sodium docecyl sulfate (SDS) which nonselectively solubilizes proteins irrespective of their native cellular location.
Nunes-Edwards, et al. (Infect. Immun. 48:492, 1985) introduced the use of radioimmunoprecipitation and cell fractionation schemes based on the use of SDS in an effort to identify leptospiral OMPs. The leptospires used in their radioimmunoprecipitation procedure were subjected to high speed centrifugation (20,000xc3x97 g) prior to the addition of antibody. Such high centrifugal forces cause mechanical disruption of the leptospiral outer membrane. Niikura, et al. (Zbl. Bakt. Hyg. A. 266:453, 1987) immunoprecipitated SDS-solubilized extracts of virulent and avirulent strains of L. interrogans serovar copenhageni that had been labeled by lactoperoxidase-catalyzed surface radioiodination. Since both of these studies precipitated a 35-36 kD doublet consistent with leptospiral endoflagella, there was a concern as to whether the other proteins identified might also have a subsurface rather than a surface location.
Jost, et al. (J. Med. Microbiol. 27:143) characterized a monoclonal antibody with specificity for a 35 kD proteinase K sensitive antigen which was present in a leptospiral outer envelope preparation. However, to demonstrate binding of the monoclonal antibody by immunoelectron microscopy, the leptospiral outer membrane had to be disrupted. Doherty, et al. (J. Med. Microbiol. 28:143) cloned two leptospiral proteins represented in an SDS-generated outer membrane preparation of L. interrogans, but did not provide corroborating evidence that these proteins are either constituents of the outer membrane or are surface-exposed.
Unsuccessful research on the identification of Leptospira and T. pallidum OMPs has shown the importance of taking into account spirochetal outer membrane fragility and the lack of outer membrane selectivity of ionic detergents such as SDS (Cunningham, et al., J.Bacteriol. 170:5789, 1988; Penn, et al., J. Gen. Microbiol. 131:2349, 1985; Stamm, et al., Infect. Immun. 55:2255, 1987). Outer membrane proteins are of great importance because they play a key role in bacterial pathogenesis. The identification of outer membrane proteins involved in Leptospira pathogenesis is significant to understanding not only leptospiral outer membrane proteins and their involvement in pathogenesis, but also to understanding other spirochetal outer membrane proteins and their role in pathogenesis.
The present invention is based on the identification of a leptospiral outer membrane protein which is associated with pathogenic strains of Leptospira. The invention describes an approximately 27.6 kD outer membrane protein, and the gene encoding the protein, originally derived from L. kirschneri. The molecular weight was calculated from the deduced amino acid sequence of the polypeptide. The amino acid sequence encodes a membrane lipoprotein with a nineteen amino acid leader peptide, a lipoprotein signal peptidase cleavage site and an amino terminal cysteine. The 27.6 kD protein has been designated ALipL32xe2x80x3 for Alipoprotein from Leptospira≅ and because the isolated polypeptide migrates, in a denaturing polyacrylamide gel, to a position corresponding to a molecular weight of 32 kD. This immunogenic polypeptide is useful for inducing an immune response to pathogenic spirochetal organisms as well as providing a diagnostic target for spirochetal-associated pathologies.
In a first embodiment, the invention provides a substantially purified LipL32 polypeptide and nucleic acid encoding the LipL32 polypeptide. In accordance with another aspect of the invention, an expression vector containing LipL32 nucleic acid is provided. Also included is a method for producing the LipL32 polypeptide.
The invention further provides a pharmaceutical composition useful for inducing an immune response to a pathogenic spirochete in a subject containing an immunologically effective amount of LipL32 in a pharmaceutically acceptable carrier.
In another aspect, the invention provides a pharmaceutical composition useful for providing immunity to a pathogenic spirochete in a subject comprising an immunogenically effective amount of antibody which binds LipL32 in a pharmaceutically acceptable carrier.
In yet another aspect, the invention provides a method for identifying a compound which binds to LipL32 polypeptide that includes incubating components comprising the compound and LipL32 polypeptide under conditions sufficient to allow the components to interact and measuring the binding of the compound to LipL32 polypeptide.
The invention further provides a method of detecting pathogenic spirochete in a sample which includes contacting a sample suspected of containing a pathogenic spirochete with a reagent that binds to the spirochete-specific cell component and detecting binding of the reagent to the component. In one aspect, the reagent that binds to the spirochete-specific cell component is an oligonucleotide probe for the identification of LipL32 nucleic acid. In another aspect, the reagent that binds to the spirochete-specific cell component is an antibody.
In another aspect, the invention provides a method for detecting antibody to LipL32 polypeptide in a sample including contacting the sample with LipL32 polypeptide, or fragments thereof, under conditions which allow the antibody to bind to LipL32 polypeptide and detecting the binding of the antibody to the LipL32 polypeptide.
In yet another aspect, the invention provides a kit useful for the detection of LipL32 polypeptide, nucleic acid encoding LipL32 and antibodies that bind to LipL32.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.